Transferring materials into cells using porous silicon

ABSTRACT

Porous and/or polycrystalline silicon are used in the delivery of substances into cells. The porous and/or polycrystalline silicon can be formed into micropiercers, microneedles and biolistic bullets for penetration of the cell. The control of the pore size and porosity of the porous and/or polycrystalline silicon allows tuning of the bioactivity of the porous silicon. The porous and/or polycrystalline silicon is also resorbable and is therefore resorbed from the cells without leaving any particles or being seen as a foreign body. Methods of manufacturing the porous silicon micropiercers, microneedles, microelectrodes, biolistic bullets, and precipitation of calcium phosphate on a bioactive substrate, and their advantages over known methods of delivering materials into cells.

CROSS REFERENCE TO RELATED APPLICATION

This application is a division of application Ser. No. 09/743,447 filedJan. 10, 2001 now U.S. Pat. No. 6,770,480, which in turn is a §371 ofPCT/GB99/02383 filed Jul. 22, 1999 which claims priority of GB9815819.9filed Jul. 22, 1998.

This invention relates to ways of transferring materials into cells, andalso to a microneedle array.

There are many times when it is necessary to transfer materials intocells, for example nucleic acids or nucleic acid constructs, such asvectors or plasmids, etc. have to be transferred into a cell for thepurposes of genetic manipulation. Furthermore, chemicals may also needto be transferred into cells, e.g. nucleotides or stains, and chemicalsto affect the physiology of a cell. A number of chemical and mechanicalprocesses have been developed to convey materials into cells. Thesetechniques include:

-   1. direct microinjection—a needle is inserted into a cell and    material expelled through the needle;-   2. electroporation the cell membrane is made permeable to some    molecules by application of a high voltage shock;-   3. biolistics—tungsten or gold particles are coated with the    substance desired to be introduced and are shot into the cell;-   4. calcium phosphate co-precipitation—cells absorb calcium    phosphate, and if DNA/other material co-precipitates with the    calcium phosphate it is also taken into the cell:-   5. mediated transformation (via liposome, viral, or bacterial    vectors): and-   6. protoplast transformation.

An aim of one aspect of the present invention is to use a new materialto assist in the transfer of substances to cells.

An aim of another aspect of the invention is to provide an improved wayof providing small volumes of a substance.

Direct microinjection involves the insertion by a microneedle of DNAdirectly into the nucleus of individual cells. A glass micropipettelinked to a micromanipulator is used to inject 10⁻⁸-10⁻⁷ μl of material,typically a solution of DNA fragments, into cell nuclei. “Hits” arealmost certain, given considerable operator expertise, but the techniqueis laborious and cannot be applied to a large number of cells.

According to a first aspect the invention comprises a method oftransferring a substance into the cell.

Preferably resorbable or bioerodable porous silicon is used.

In one embodiment a microneedle that comprises at least a region ofporous silicon is used or an array of such microneedles is used.

According to a second aspect the invention comprises a microneedle (ormicroneedle array) comprising porous silicon.

According to a third aspect the invention comprises a vehicle fortransferring material into a cell, the vehicle comprising, at least inpart, porous silicon and material to be transferred into the cell.

Preferably the porous silicon is resorbable. The vehicle may comprise aporous silicon biolistic bullet. The vehicle may comprise a substancewhich in use will co-precipitate with a co-precipitate substance that istaken into the cell. The vehicle may comprise an electrically-conductingbioactive porous silicon electrode.

According to a fourth aspect the invention comprises the use of poroussilicon as a transfer medium for transferring materials into a livingcell.

It has been discovered that porous silicon is biocompatible, and it hasnow been discovered that porous silicon can be corroded in, or resorbedinto, a mammalian body without significant detrimental effect. Poroussilicon can be used to locate and substantially immobilise biologicalmaterial (or any substance to be introduced into a cell), with thesubstance being free enough once in the cell to combine with cell DNA,or otherwise be released to have an effect.

It is known from PCT Patent Application No. WO 96/10630 to have an arrayof micromachined bulk silicon barbs or tips and to use them mechanicallyto pierce the plasma membrane of large numbers of cells simultaneously.This is more efficient than piercing a single cell with a single needle,which can result in a laborious operation if hundreds of cells need tohave material introduced into them. The tips of WO 96/10630 are, withhindsight, less effective at transferring material (e.g. DNA) into apierced cell than they might be. It is, for example, proposed in thatdocument to use surface tension forces between closely-spaced tips tohold biological material to be introduced into the cells in the spacesbetween the tips, and to trap it between the tips (probes) and thesubstrate.

A proposal is discussed in U.S. Pat. No. 5,262,128 which was publishedin 1993 and purports to teach the man skilled in the art to make anarray of silicon needles using the Liga Processes. It is believed thatthis document is non-enabling at its filing (and publication) date andis not prejudicial to the novelty of the present invention for thatreason. In 1989 when the application was filed, and in 1993 when it waspublished, the skilled man of ordinary expertise could not make verythin silicon needles having a central lumen as discussed in the documentusing the techniques discussed. The Liga Process is not suitable formanufacturing hollow needles in silicon and does not enable slopingstructures to be made.

U.S. Pat. No. 5,457,041 discloses an array of solid needles made ofsilicon having ragged tips.

U.S. Pat. No. 5,591,139 discloses a silicon microneedle that is formedin the plane of a silicon wafer.

WO 97/06101 discloses a method for producing bioactive silicon as awafer, and suggests uses for bioactive silicon in the fabrication ofbiosensors and in bioassays.

WO 92/01802 discloses the idea of gem substance into a cell byincorporating the substance in a liquid and making the particles of theliquid, and then accelerating the ice particles to penetrate the cells,the ice particles melting after cell-penetration.

JP 06 034 361 discloses a porous silicon atomic force microscope tip.The device does not penetrate the sumacs being imaged.

U.S. Pat. No. 4,969,468 discusses solid metal needles for electricalcontact with nerves.

According to another aspect tab invention comprises a cell-penetratingmember or micropiercer made of porous silicon.

The cell-penetrating member is adapted to have a substance to beintroduced into a cell carried by to porous silicon.

According to another aspect, the invention comprises a cell penetratingmember or a micropiercer comprising at least a region of porous silicon.Preferably the substance comprises DNA or RNA, a fragment of DNA or RNA,or a construct of DNA or RNA.

The cell penetrating member or micropiercer is preferably adapted tohave a substance to be introduced to a cell carried by porous silicon.

The porous silicon region is adapted to immobilise a substance (e.g.DNA) in comparison with its mobility when provided with a bioinertsubstance such as titanium. The porous silicon region is preferably atthe tip of the cell penetrating member or micropiercer. The cellpenetrating member or micropiercer may be a tip or barb, with no centrallumen or it may be a needle with a central channel. The cell gene-ratingmember or micropiercer may have a capilliary or pore network extendingfrom a reservoir or channel to a substance delivery region provided onthe surface of the cell penetrating member or micropiercer.

The cell penetrating member or micropiercer may have a coating of poroussilicon or it may be porous throughout its cross-section, at least atits tip (or other substance delivery region if that is not the tip).Substantially the whole exterior surface of the cell penetrating memberor micropiercer that penetrates a cell in use may comprise poroussilicon. The cell penetrating member or micropiercer may be a bulksilicon microtip with a porous silicon coating.

An advantage of holding the substance to be introduced to the cell atthe tip of the cell penetrating member or micropiercer itself, insteadof in channels/spaces between tips, is that the material is definitelyintroduced into the cell, and typically deeply into the cell. This mayincrease the success rate of the operation (in many cases introducingDNA into cells and stable uptake of the DNA/fragment is notstatistically very successful—a few percent may succeed, which is why somany cells have to be injected).

Instead of using porous silicon to immobilise the material on thetip/ensure at least some material is present on the tip, other holdingmeans may be used. For example, polycrystalline silicon can hold somesubstances at grain boundaries. The holding means may comprise a porousmaterial.

It is known to immobilise DNA fragments in macroporous silicon in thefield of a flow-through genosensor (Advances in Genosensor Research. K.L. Beattie et al. Clin. Chem. 41, 700 (1995)).

An advantage of porous silicon is that its bioactivity can be tuned bycontrolling its pore size and porosity. It is therefore possible tocreate a cell penetrating member or micropiercer with a porous tip withpores tailored to hold/immobilise a particular desired molecule orsubstance. Of course, the substance will not be so immobilised that atleast some of the material cannot leave the tip when the tip is in thecell.

Porous silicon has another great advantage as the choice of material fora cell penetrating member or micropiercer in that micromachiningtechniques for fabricating small scale devices from silicon exist, e.g.in the electronics industry.

It is known how to make a silicon structure porous (see for example U.S.Pat. No. 5,348,618).

An array of cell penetrating members or micropiercers may be provided.The array is preferably a two-dimensional array of n×m micropiercers.The micropiercers are preferably regularly disposed in a grid pattern,but they perhaps do not need to be.

It is also known to have an array of microtips for a completelydifferent purpose—for field emission cathodes used in vacuummicroelectronic applications. Here, a 5 mm square silicon chip willtypically contain about 500 microtips of pyramidal shape with tip)widths of 50 mm-1 μm and heights of 10-100 μm, depending upon themanufacturing parameters chosen. With hindsight, these would be suitablefor porosification and then use as micropiercers for transferring asubstance into cells. It is also even known to haste porous siliconpyramidal cathodes—e.g. Field emission from, pyramidal cathodes coveredin porous silicon. P. R. Wilshaw et al. J. Vac. Sci. Techn. B12,1(1994); Fabrication of Si field emitters by forming porous silicon. D.Kim et al. J. Vac. Sci. Tech. B14, 1906 (1996); and Porous silicon fieldemission cathode development. J. R. Jessing et al. J. Vac. Sci. Techn.B14, 1899 (1996). However, these are all in a totally different field,and one show a micropiercer having held on it DNA, RNA or any othersubstance to be introduced into a cell.

According to a third aspect, the invention comprises a method ofproducing a micropiercer device comprising manufacturing one or moremicropiercer projections, and providing substance holding means at ornear the tip of the projections.

Preferably the method comprises making at least a part of theprojections porous. Preferably the method comprises making the tip ofthe projections porous or substantially the entire extent of the tipsporous, or providing a porous coating on the tip. Preferably the tip ismade porous using an HF anodising technique.

According to another aspect, the invention comprises a method oftransferring a material into a cell comprising associating the materialwith a tip portion of a micropiercer and piercing the cell with themicropiercer.

Preferably the method comprises using porous silicon to locate thematerial at or near the tip portion.

According to a further aspect, the invention comprises a method ofgenetic manipulation of a cell comprising associating genetic materialwith a tip portion of a micropiercer, piercing the cell with themicropiercer to allow the genetic material to enter the cell. Thegenetic material may then be stably incorporated in the cell.

According to another aspect, the invention comprises a microneedle arraycomprising a plurality of needles extending away from a support, theneedles each having fluid transport means adapted to transport fluidfrom their bases to their tips, and fluid supply means communicatingwith the fluid transport means and adapted to supply fluid to beinjected to the base of the needles.

Preferably the array of microneedles are made of silicon. It may bemicromachined, for example from a silicon wafer.

The fluid transport means may comprise a reservoir, which may extendunder the needles. The support may have a lower portion, an upperportion, and a channel or reservoir extending between the upper andlower portions, with the needles being provided in the upper portion andthe fluid transport means extending to the reservoir or channel.

The fluid transport means may comprise a lumen, or macropore in eachneedle which may extend generally centrally of the needle through itslongitudinal extent. Alternatively, or additionally, the fluid transportmeans may comprise a pore or capillary network, such as a plurality ofmesopores.

The array of needles may be provided on an integrated silicon chip,which may also have a sensor provided on it, the sensor preferablyenabling one to monitor in situ the transfection process. For example aphoto emitter/detector may be used in association with light emittingmarkers (e.g. fluorescent) associated with the DNA. It may also bedesirable to have a power supply and/or processing circuitry, and/orcontrol circuitry provided on the chip. Arrays of light emitting devisesand photodetectors may enable the transfection process to be monitoredunder high-spatial resolution.

According to another aspect the invention comprises a method ofmanufacturing a microneedle, or a microneedle array, the methodcomprising taking a bulk silicon wafer and creating a needle or an arrayof needles; and creating fluid transfer means extending from the base ofthe or each needle to its tip.

Preferably, the method comprises providing a network of pores from thebase of the or each needle to its tip. The pores may be macropores ormesopores, or for some applications they may even be micropores (butmacropores are preferred).

The or each needle may be created using photolithographic techniquessuch as anisotropic etching and photo-resist lithographic techniques.

The silicon substrate may be an n-type substrate with a resistivity inthe range of 0.1-10 Ωcm.

The needle or needle array may be planarised, for example by use of anon-conducting mask.

The planarised array may then be treated so as to expose just the tips,for example by using an oxygen plasma treatment and an HF dip to exposethe tips alone. The planarised array may also be in filled. The tips canthen be anodised to create the pores from the tip to the wafer backsurface. The wafer, provided with an array of tips, may then be bondedto another backing member, which may be shaped so as to define a channelor reservoir between the tip-carrying wafer and the backing member.

According to a further aspect the invention comprises a vehicle fortransferring material into a cell, the vehicle comprising at least inpart resorbable material.

Preferably the vehicle comprises resorbable silicon, such as poroussilicon, or polycrystalline silicon. The whole of the vehicle may bemade of the resorbable material, or only part of it. The vehicle maycomprise bioactive silicon. (By “resorbable” it is meant that thematerial is corroded/absorbed/eroded/or otherwise disappears when insitu in physiological fluids. By “bioactive” it is meant that thematerial can induce the deposition of calcium phosphate precipitates onits surface under physiological conditions (when in body fluids)).

If the vehicle is retained in the cell it will beadsorbed/corroded/eroded or resorbed, or partially resorbed, and be lessof an irritation/foreign body to the cell in due course.

The resorbable silicon/other material may be used in a biolisticstechnique. For example the vehicle for transferring material into a cellmay be a biolistic bullet.

The vehicle for transferring material into the cell may comprise abiolistic bullet comprising porous silicon.

The bullet may have a substance to be introduced into a cell adhered toit. The bullet may be impregnated with material (e.g. DNA material). Itmay be substantially saturated with material. The bullet may comprise asubmicron silicon particle. The silicon particle may be rendered porousby stain etching techniques. The particle is preferably mesoporous.

A resorbable biolistic bullet would not leave behind in the cell aparticle, as do gold or tungsten biolistic bullets. The bullet need notbe porous all of the way through—it may have a porous coating. Theresorbable bullet need not necessarily be made of porous silicon, or ofsilicon at all, but porous silicon has been identified as an especiallysuitable material.

According to another aspect, the invention provides a method oftransferring material into a cell comprising the steps of shooting avehicle carrying said material into the cell.

Preferably the vehicle is the vehicle as hereinabove defined. Preferablythe bullet is shot into the cell be means of a pressurised gas, forexample helium.

The process of biological biolistics is often used where more standardtechniques do not work. Resorbable impregnated materials, such as poroussilicon offer biocompatible advantages over corrosion-resistant bulkmetal materials.

According to a further aspect the present invention provides a method ofmaking a vehicle for transferring material into a cell comprising thesteps of rendering the vehicle at least partially porous and introducingto the vehicle the material to be transferred to the cell.

Preferably the vehicle comprises a silicon bullet, most preferably asubmicron silicon particle, which may be rendered porous, preferablymesoporous by stain etching techniques. The bullet may have the materialto be introduced to the cell, adhered to it or alternatively it may beimpregnated with the material.

The vehicle may be a submicron particle and the material to betransferred to the cell may be co-precipitated (with a precipitatesubstance) using the vehicle as a nucleation site.

The vehicle for transferring material into a cell may comprise bioactivesilicon. The vehicle may have associated with it material to betransferred in a form adapted to co-precipitate with a substance whichis taken up by cells. The co-precipitate may be a calcium phosphateprecipitate.

According to another aspect the invention comprises a method ofintroducing material into a cell comprising associating the materialwith a silicon particle, precipitating calcium phosphate onto theparticle to form a calcium phosphate/silicon particle combined particle,and arranging for the cell to uptake the calcium phosphate/siliconparticle combination.

In the technique of electroporation the cell membrane can be madepermeable by exposing cells to a brief electric shock of very highvoltage. Low porosity bioactive silicon is electrically conducting andis suitably developed as an intimate coupling matrix for adherentmammalian cells growing on microelectrode arrays.

By having bioactive silicon, e.g. porous silicon or polycrystallinesilicon as one or both electrodes in electroporation apparatus it isenvisaged that better DNA transfer takes place.

According to a further aspect the invention comprises a method ofelectroporation comprising providing an electrically conductingbioactive silicon electrode.

Preferably the method comprises growing cells on the electrode. Themethod may comprise providing an array of bioactive silicon electrodes,possibly with cells grown on them. The electrode, or electrodes, may becoated with porous silicon or may be of porous silicon throughout theircross-section, at least at a region of their height.

According to a further aspect the invention comprises electroporosisapparatus comprising a bioactive electrode. Preferably the electrode isbioactive silicon, most preferably porous silicon. An array ofelectrodes, or microelectrodes, may be provided.

The invention may also reside in the use of bioactive silicon,preferably porous silicon, in the preparation of apparatus for theintroduction of materials into cells.

It may be helpful to clarify that bioactive materials are a class ofmaterials which when in vivo elicit a specific biological response thatresults in the formation of a bond between living tissue and thatmaterial. Bioactive materials are also referred to as surface reactivebiomaterials. Resorbable materials are materials which are designed todisappear or degrade gradually over time in vivo, and they may or maynot be replaced with tissue. Bioerodable materials are materials whicherode in vivo, with the material possibly being absorbed by cells, orpossibly not being absorbed. A bioinert material is a material thatelicits no local gross biological response in vivo.

BRIEF DESCRIPTION OF THE DRAWINGS

Several embodiments of the present invention will now be described bymeans of example only with reference to the Figures, in which:

FIG. 1 shows a partially porosified silicon microtip;

FIG. 2 shows a silicon microneedle array;

FIG. 3 shows the silicon microneedle array of FIG. 2 having amacroporous network running from the tip to an underlying reservoir;

FIG. 4 shows a porous silicon bullet impregnated with DNA;

FIG. 5 shows a porous silicon core impregnated with DNA and surroundedby calcium phosphate;

FIG. 6 illustrates the electroporation technique of the presentinvention; and

FIGS. 7 and 8 show SIMS plots demonstrating the affinity of DNA forporous silicon and that it can be released from a porous siliconsurface.

FIG. 1 shows a micropiercer 10 in the form of a microtip 12 having abase width A of 50 μm and a height B of 100 μm and a tip width C of 0.5μm. The surface of the microtip 12 is coated with porous silicon 14having a depth D of 0.1 μm.

In use, the porous silicon coating 14 immobilises the substance to bedelivered to the cell (e.g. DNA/RNA) on the tip itself, which increasesthe chances of the immobilised substance on the tip being introducedinto the living cell.

The pore size and porosity of the porous silicon coating can becontrolled to tune the bioactivity of the microtip 12. By controllingthe pore size and porosity of the porous silicon, we can make particularmolecules come off it more, or less, readily. We may leave the microtipsinside a cell for a predetermined time to allow molecules todisassociate themselves from the porous silicon.

FIG. 2 shows an array 20 of silicon microneedles 22 extending away froma silicon support, or back, member 24. The microneedles 22 have poroussilicon microtips 26 and a central lumen 28 communicating between themicrotips 26 and a reservoir 30 defined between an upper member 32,provided with the microneedles 22 and the back, support, member 24. Theback member 24 is of bulk silicon.

FIG. 3 shows an array 33 of silicon microneedles 34 that is similar tothat of FIG. 2. The principal difference between the arrays shown inFIGS. 2 and 3 is that the microneedles 34 shown in FIG. 3 are notprovided with a central lumen 28. Instead the array 33 of siliconmicroneedles 34 in FIG. 3 is provided with a mesoporous network 36 whichextends from the microtips of the microneedles to the reservoir 30′,allowing fluid communication between the reservoir 30′ and themicrotips.

In use, the substance to be delivered to cells is provided to the poroussilicon microtips 22,34 from the reservoir 30,30′ through the centrallumens 28 or the mesoporous network 36. The substance is then held bythe porous silicon microtips ready for introduction into a cell.

The material to be introduced into the cells may be pumped into thereservoir 30, 30′, and out through the lumens 28 or porous network by apump, not shown (but arrow 39 indicates the pump delivering liquid tothe reservoir).

All or part of the silicon surfaces within the final structure may betreated in such a way as to modify their interaction with biologicalsystems. This might be achieved by forming a layer of porous silicon onthe surface. Such a layer could be formed by either an electrochemicalanodisation process or possibly by immersing the structure into a stainetching solution such as a mixture of hydrofluoric acid and nitric acid.

FIG. 4 shows a biolistic bullet 40 comprising a submicron siliconparticle rendered mesoporous by stain etching.

In use, the bullet 40 is impregnated with the substance to be introducedinto a cell and is shot into the cell using pressurised helium. As theporous silicon is a resorbable material, it will be preferably fullyresorbed, and at least partially resorbed, by the cell that it entered,and thus comprises less of a foreign body than known biolistic bulletssuch as gold or tungsten which leave particles of metal in the cell.

FIG. 5 shows a porous silicon core 50 impregnated with a substance to beintroduced into a cell (e.g. DNA/RNA) and calcium phosphate precipitate52 formed around the core 50. The calcium phosphate 52 isco-precipitated with DNA/RNA, so that a genetic material/calciumphosphate layer surrounds the bioactive silicon core 50.

The bioactive silicon core locally induces calcium phosphatesupersaturisation. It may be possible to place a bioactive silicon corenext to a cell/against the wall of a cell, and co-precipitateDNA/Ca(PO₄)₂ against the core and against the wall of the cell. If thecore is phagocytosed it can be resorbed.

The core 50 need not have DNA/RNA/any active substance on it—it maysimply serve as a good nucleation site for co-precipitation of DNA/Ca(PO₄)₂.

It is known to use glass beads as a nucleation site for calciumphosphate co-precipitation DNA transfection—see for example the paper byWatson and Latchman in “Methods (San Diego) 1996 10(3), 289-291 (Eng).

It will be appreciated that microprobes are pores with a diameter of 2nm or less; mesopores have a diameter of 2 nm-50 nm; and macropores havea diameter of 50 nm or more.

It has also been realised that it is possible to improve the efficiencyof the introduction of materials to cells in an electroporationtechnique, as shown in FIG. 6, using porous silicon, preferablymesoporous silicon (but macroporous and microporous silicon are alsouseful).

The use of a porous silicon (or porous other bioactive material, orbioactive polycrystalline silicon) electrode 60,61 achieves betterperformance in electroporation. Because the electrode is bioactive,instead of being bioinert cells (typically animal cells) have anaffinity to it and are localised on its surface.

Low porosity (50% or less, or 30% or less, or 10% or less) bioactivesilicon is electrically conducting and is a suitable intimate complexmatrix for adherent mammalian cells 62, which may grow on amicroelectrode array 60,61. Thus, it is possible to grow mammalian cellson bioactive porous silicon electrodes and then introduce DNA (or othersubstances) into the cells by using electroporation, with the substrateupon which the cells are grown being an electrode, or even bothelectrodes 60,61, of the electroporation apparatus. This has advantagesin handling the cells, and achieves a better efficiency rate of DNAintroduction than solely having the cells suspended in a liquid medium63.

The fact that porous silicon is resorbable/erodable in vivo in mammalshas been proved by the inventors, and this underpins some aspects of theinvention. The fact that silicon can be made bioactive underpins otheraspects of the invention.

FIG. 7 shows a SIMS plot (Secondary Ion Mass Spectroscopy) showing theconcentration of nitrogen with depth in a sheet of porous silicon. DNAis rich in nitrogen, and detecting high nitrogen levels in the poroussilicon is a measure of how much DNA is present. Plot 70 shows the“aged” porous silicon sheet analysed for nitrogen, with no DNA added tothe surface of the sheet. The background level of nitrogen depends onthe type of porous silicon film and its “age”—the duration of storage inambient air. Plot 72 shows the analysis of the porous silicon sheetafter a single drop of water has been applied to the surface of theporous silicon sheet. There was 1 ng per μ litre of DNA in the drop ofwater. The DNA solution drop was dried at 50° C. before the sheet wasanalysed. Plot 74 shows the amount of nitrogen in the porous siliconwhen the same 1 ng per μ litre of DNA in water drop is applied to thesheet and dried, and then the sheet is washed in deionised water at 50°C.

As will be seen, there is far more nitrogen shown in plot 72 than inplot 70, showing that the DNA is being detected by the test. Plot 74shows that the washing step removed some, but not all, of the DNA—thatsome of the DNA was probably partially immobilised on the poroussilicon, to be released later (during washing).

FIG. 8 shows equivalent SIMS plots for the same layer after pure watertreatment. “Aged” porous silicon has been stored in ambient air and hasacquired a background level of nitrogen due to adsorption of nitrousoxides and ammonia—common trace pollutant gases. Plot 80 shows agedporous silicon with no DNA, plot 82 aged porous silicon with a waterdroplet deposit (no DNA solution), and plot 84 shows again forcomparison the analysis of aged porous silicon with 1 ng/μ litre of DNAin solution added and dried at 50° C.

The SIMS data shown in FIGS. 7 and 8 demonstrate that porous silicon canreversibly bind DNA.

The invention can perhaps be thought of as using porous silicon (orperhaps polycrystalline silicon) as an inorganic vector fortransporting/transferring material into a living cell.

1. A method of transferring a substance into a cell comprising using asub-micron silicon particle comprising silicon for conveying thesubstance into the cell, the particle comprising a porous surface layer.2. A method according to claim 1 wherein the method comprises the stepof transferring at least part of the silicon into the cell.
 3. A methodaccording to claim 1 wherein the porous surface layer is obtainable by astain etch process.